In the majority of mammals there are proteins which have anti-coagulant properties. These proteins can be divided, as follows, into three groups based on their different mechanisms of activity:
1. Proteins which form a complex with the coagulating factor and thereby render the coagulating factor ineffective. These include the following proteins: PA0 2. Proteins which proteolytically cut up a coagulation factor and thereby inactivate the coagulation factor. The only protein of this kind which has hitherto been described is protein C (J. Biol. Chem. 251:355-363 (1976)). PA0 3. proteins which screen off and/or hydrolyse the negatively charged phospholipids, so that the phospholipid-dependent reactions of the coagulation mechanism are inhibited. Hitherto, only phospholipases isolated from various types of snake venom have been described (Eur. J. Biochem 112:25-32 (1980)). PA0 1. The serine protease factor Xa and thrombin are inactivated as a result of binding to anti-thrombin III or to the anti-thrombin/heparin complex. Both the pro-thrombin activation and also the formation of fibrin can be inhibited in this way. In addition to anti-thrombin III, there are various other plasma-protease inhibitors such as, for example, .alpha.2 macroglobulin and anti-trypsin, the effect of which is dependent on time. PA0 2. The discovering of protein C resulted in the discovery of another anti-coagulation mechanism. Once protein C has been activated, it acts as an anti-coagulant by selective proteolysis of the protein co-factors factor Va and VIIIa, by means of which prothrombinase and the enzyme which reacts with factor X are inactivated. PA0 3. Plasmin cleaves monomeric fibrin 1, a product of the action of thrombin on fibrinogen, thereby preventing the formation of an insoluble fibrin (Nosssel, H. L., Nature 291: 165-167 (1981)).
a) Anti-thrombin-III (Thromb. Res. 5:439-452 (1974)) PA1 b) .alpha.-Protease Inhibitor (Ann. Rev. Biochem. 52: 655-709 (1983)) PA1 c) .alpha.2-Macroglobulin (Ann. Rev. Biochem. 52:655-709 (1983)) PA1 d) C1-Inhibitor (Biochemistry 20:2738-2743 (1981)) PA1 e) Protease Nexin (J. Biol. Chem. 258:10439-10444 (1983)). PA1 a) obtaining cells which express an annexine, PA1 b) homogenizing said cells and adjusting the pH to about 8.0 to about 10.0, PA1 c) adding at least one bivalent cation selected from the group Ca.sup.2+, Cd.sup.2+, Zn.sup.2+, Mn.sup.2+ and Co.sup.2+, PA1 d) adding a phospholipid, PA1 e) washing the insoluble cell residue to remove the soluble constituents, and PA1 f) extracting the annexine from the cell residue with a chelating agent. PA1 a) obtaining cells which express a VAC, PA1 b) inactivating and homogenizing said cells and adjusting the pH to about 9.0, PA1 c) adding Ca.sup.2+, PA1 d) adding lecithin, PA1 e) washing the insoluble cell residue to remove the soluble constituents, and PA1 f) extracting the VAC from the cell residue with EDTA.
The coagulation system, which proceeds step by step, has been intensively investigated in recent years. It is understood to be a self-intensifying multi-step system of various interconnected proteolytic reactions in which one enzyme converts a zymogen into the active form (cf. Jackson C. M., Nemerson Y., Ann. Rev. Biochem. 49:765-811 (1980)). The speed of this reaction is critically increased by the presence of phospholipids and other co-factors such as factor Va and factor VIIIa. In vivo, the pro-coagulation reactions are regulated by various inhibiting mechanisms which prevent an explosively thrombotic trauma after slight activation of the coagulation cascade.
The anti-coagulation mechanism can be sub-divided as follows (Rosenberg, R. D., Rosenberg, J. S., J. Clin. Invest. 74:1-6 (1984)):
Of the native proteins involved in the coagulation process mentioned above, only anti-thrombin III is currently in clinical use. However, a serious disadvantage of the use of this protein is the increased tendency to bleeding.
All of the agents hitherto used as anti-coagulants, be they native or synthetic by nature, render the coagulation factors inactive in some way and thereby produce side effects which may have a disadvantageous effect on the coagulation process.
Surprisingly, in addition to these proteins, other native substances have been isolated which still show the desired anti-coagulant properties under particular conditions but do not increase the risk of bleeding. In the case of major bleeding, these proteins lose their anti-coagulant properties and consequently, in such cases, their use does not interfere with the coagulation processes necessary for survival. As they were first isolated from strongly vascularized tissue they are known as vascular anti-coagulating proteins, VAC.
The proteins isolated from strongly vascularized tissues such as umbilical cord vessels and placenta have molecular weights of about 70.times.103, about 60.times.103, about 34.times.103 and about 32.times.103, of which the substances with the molecular weights of 34 and 32.times.103 consist of a single polypeptide chain. The precise biochemical characterization of these proteins and the methods for isolating and purifying them can be found in EP-A-0 181 465, which is incorporated by reference herein in its entirety.
Proteins with a VAC activity are natural blood coagulation inhibitors which interfere with the blood coagulation cascade at two points. The first time, they inhibit the activation of factor X into Xa, catalyzed by factors IXa and VIIIa, and the second time they suppress the cleaving of prothrombin to form thrombin, which is mediated by factors Xa and Va. One fact common to both activation steps is that they require calcium ions and phospholipids. Obviously, VAC proteins are also able to interact with phospholipids and, as a result of this binding, block the activation steps of the coagulating factors.
It has been found in the meantime that there is a whole family of substances which, like VAC, bind to phospholipids in a calcium-dependent manner and interfere with processes dependent on phospholipid surfaces. This family, which is also known as the annexines, includes not only lipocortin I, calpactin I, protein II, lipocortin III, p67-calelectrin but also IBC, PAP, PAP I, PP4, endonexin II and lipocortin V.
The common structural features of the annexines probably form the basis for their similar Ca2+ and phospholipid binding properties. Although this general property is true of all annexines, there is a clear individuality with regard to their affinity for Ca2+ and the various types of phospholipid.
The physiological functions of the annexines are concerned with membrane-associated processes. The basic mechanism of the anti-coagulant effect of VAC has been recognized as the inhibition of the catalytic capacity of phospholipids by binding VAC to the surface thereof, thus preventing the formation of the coagulation-promoting complex on the surface.
Other annexines are also capable of inhibiting coagulation, but VAC appears to be the most effective inhibitor.
Binding studies have shown that VAC associates reversibly with pro-coagulatory phospholipids, in calcium dependent manner. Other bivalent cations selected from the series Cd2+, Zn2+, Mn2+ and Co2+ also have a positive effect on association, but not to the same extent as Ca2+.
These properties make the proteins interesting and extremely valuable active substances from a pharmacological point of view. The genetic engineering method which made it possible to produce VAC proteins is described in EPA 293 567, the disclosure of which is fully incorporated by reference herein.
In the process disclosed in EPA 293 567, the frozen biomass was suspended in a suitable lysing buffer in order to isolate and purify the expressed proteins. The cells were then mechanically destroyed, for example, using a Manton-Gaulin press. After the addition of a precipitating agent for non-protein constituents such as polyethylenimine, the solid constituents were removed, for example, by centrifugation. After precipitation of the proteins, preferably by ammonium sulphate fractionation, dissolving the precipitate, removing the precipitating agent and clarifying the solution, the extract thus obtained was subjected to various chromatographic purification procedures. Instead of precipitation of proteins, the crude VAC extract can also be purified by chromatographic pre-purification provided that it can then be subjected to a later cleaning cycle. SiO.sub.2 has proved suitable as the column material for the pre-purification, for example, but other materials with similar properties are also suitable. According to the invention, silica catalyst carrier grade 953 W made by Messrs Grace was used.
A chromatographic purification cycle suitable for purifying the proteins according to the invention consisted, for example, of a DEAE-Fast-Flow-Sepharose, a sephacryl S-200 high resolution and a Q-Sepharose-Fast-Flow-Chromatography. The purity of the proteins according to the invention obtained in this way was determined by SDS-PAGE, western blot, gel permeation HPLC, reverse HPLC and isoelectric focusing.